Immunofluorescence Cell Counting and Analysis in Fiji v1

Objective: To quantify specific starter populations of AAV infected dopamine (DA) neuronswithin the Substantia Nigra pars compacta (SNc) or Ventral Tegmental Area (VTA)in multi-channel immunofluorescence images of brain sections. Experimental Model System: This study utilizes a dualrecombinase-based strategy to genetically target and label distinctsubpopulations of midbrain dopamine (DA) neurons. To label a specific DA neuronsubtype, we employed a transgenic mouse model expressing Cre recombinase undera subtype-specific promoter and FlpO recombinase under the dopamine transporter(DAT) promoter. These animals were stereotactically injected with a CONFON(Cre-ON/FlpO-ON) virus driving the expression of eYFP, which labels thetargeted subtype in green. Concurrently, a COFFON (Cre-OFF/FlpO-ON) virusdriving mCherry expression was co-injected. This virus labels the broaderpopulation of DAT-expressing DA neurons that are not part of the specificCre-expressing subtype in red. This approach results in the following labeling: Green (eYFP): DA neurons of the specific genetic subtype. Red (mCherry): Other DA neurons within the injection site vicinity. To confirm dopaminergic identity, dopamine neurons are also immunostained with Tyrosine Hydroxylase (TH) with a Far-red labelled secondary antibody such as Alexa fluor 647. Nuclei are labelled with DAPI. Quantification of these starter populations within the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) is critical for interpreting subsequent axonal staining patterns in downstream target regions. The protocol below details the method for quantifying these DA neuron populations through image adjustment, application of standardized regions of interest (ROIs), and manual cell counting based on strict criteria. Video and image document provided